Desarrollo, evaluación y aplicación de marcadores moleculares para el análisis genético de la coquina "Donax truculus"

[Resumen] La coquina Donax trunculus es un importante recurso marisquero en la Península Ibérica que se encuentra en regresión en los bancos naturales gallegos. Desafortunadamente, no existen trabajos sobre variación genética en y entre poblaciones, necesarios para desarrollar estrategias de conservación, gestión y recuperación. En este trabajo se estudió la diversidad y estructura poblacional de bancos naturales de la Península Ibérica mediante PCRs multiplex de microsatélites y marcadores de ADN mitocondrial (citocromo B y COI). También se usaron microsatélites y análisis de paternidad para evaluar la obtención de semilla en criadero y el posible impacto de su empleo en repoblación. Por último, se evaluaron PCRRFLPs del gen mitocondrial COI para identificar cuatro especies del género Donax. La evaluación poblacional reveló la existencia de diferenciación genética entre Atlántico y Mediterráneo. El análisis de paternidad mostró un descenso moderado del tamaño efectivo esperado de los reproductores y un incremento ligero de la endogamia (< 5%), lo que evitaría la pérdida de alelos a baja frecuencia. Además, se observó diferenciación genética entre reproductores y dos grupos de semilla (T2 y T4), por lo que su empleo en repoblación podría alterar la estructura genética de los bancos naturales. Tres endonucleasas mostraron patrones de restricción para el gen COI que permitieron discriminar entre D. semistriatus, D. trunculus, D. variegatus y D. vittatus.[Resumo] A cadelucha Donax trunculus é un importante recurso marisqueiro na Península Ibérica que se atopa en regresión nos bancos naturais galegos. Desafortunadamente, os traballos sobre variación xenética en e entre poboacións, precisos para levar a cabo estratexias de conservación, xestión e recuperación, son inexistentes. Neste traballo estudouse a diversidade e estrutura poboacional de bancos naturais da Península Ibérica mediante PCRs multiplex de microsatélites e marcadores de ADN mitocondrial (citocromo B e COI). Tamén se empregaron microsatélites para, mediante análisis de paternidade, avaliar a obtención de semente en criadeiro así como o posible impacto do seu emprego en repoboación. Por último, avaliáronse PCR-RFLPs do xene mitocondrial COI para a identificación de catro especies do xénero Donax. A avaliación poboacional revelou a existencia de diferenciación xenética entre Atlántico e Mediterráneo. A análise de paternidade mostrou un descenso moderado do tamaño efectivo esperado dos reproductores e un incremento lixeiro da endogamia (<5%), o que evitaría a pérdida de alelos a baixa frecuencia. Ademais, observouse diferenciación xenética entre reprodutores e dous grupos de semente (T2 e T4), polo que o seu emprego na repoboación podería alterar a estrutura xenética dos bancos naturais. Tres endonucleasas mostraron patróns de restricción para o xene COI que permitiron discriminar entre D. semistriatus, D. trunculus, D. variegatus e D. vittatus.[Abstract] The wedge clam Donax trunculus is an important shellfish resource in the Iberian Peninsula that it is in regression in Galician natural beds. Unfortunately, studies about genetic variation in and between populations, which are necessary to develop conservation, management and restoration strategies, are nonexistent. In this study microsatellite multiplex PCRs and mitochondrial markers (Cytochrome B and COI) were used to analyze the population diversity and differentiation of natural beds from Iberian Peninsula. Paternity assignment was also carried out in hatchery seed with microsatellites. This was used to evaluate genetic changes introduced through hatchery production and to asses the potential impact of using this seed in restocking. Finally, PCR-RFLPs of mitochondrial COI gene were evaluated to distinguish four Donax species. Population analysis displayed the existence of genetic differentiation between Atlantic and Mediterranean. Parentage analysis showed a moderate reduction of the breeders’ expected effective population size and a slight increase of inbreeding (<5%), which would allow to preserve low frequency alleles. Moreover, genetic differentiation was observed between breeders and two groups of seed (T2 and T4). Its use in restoration could change the genetic structure of natural populations. Three endonucleases showed restriction patterns of COI gene that allowed to distinguish between D. semistriatus, D. trunculus, D. variegatus and D. vittatus

Genetic Variability in Ruditapes Decussatus Clam Combined With Perkinsus Infection Level to Support Founder Population Selection for a Breeding Program

[Abstract] Clam farmers worldwide face several challenges, including irregular seed supply and high mortalities due to pathogenic organisms such as "Perkinsus olseni". In Europe, there is a high unmet consumer demand for native clam species such as "Ruditapes decussatus". The high market value of "R. decussatus" makes the culture of this species potentially more attractive than that culture of the alien species "Ruditapes philippinarum". Thus, there is a market opportunity in breeding and producing "R. decussatus" at an industrial scale. A selective breeding program to improve "R. decussatus" performance will be carried out in Portugal; and the first critical step to develop such a breeding program is the establishment of a founder population. In this study, intra- and interpopulation genetic diversity was assessed using 13 microsatellite markers in eight natural beds located in Portugal, Spain and Italy. Also, allele and genotypic frequencies of each microsatellite locus were assessed discriminating between clams infected and non-infected by "P. olseni". All locations showed similar values for several genetic diversity parameters. Analyses of population differentiation (FST, Bayesian clustering and AMOVAs) revealed five genetically differentiated regions: Rías Altas and Rías Baixas (NW Spain), North/Central Coast of Portugal, Gulf of Cadiz and Adriatic Sea. Significant differences in the allelic and genotypic frequency distribution between infected clams and non-infected ones at four microsatellite loci are reported suggesting that resistance to the disease could have a genetic basis. Moreover, a positive or negative relationship between the frequency of certain alleles and the parasite infection was inferred. Further studies should confirm the potential use of those alleles as genetic markers for "P. olseni" infection. Integrating results of genetic diversity within and between populations and Perkinsus infection levels, a founder population for a "R. decussatu" breeding program is proposed, composed by individuals from Barallobre (Rías Altas), Pontevedra or Cangas (Rías Baixas), Óbidos (North/Central Coast of Portugal), Algarve (Gulf of Cadiz) and Venice (Adriatic Sea).Governo da República Portuguesa; 22390 (16/SI/2016) (CENTRO-01-0247-FEDER-022390)Ministério do Mar (Portugal); FA_01_2017_00

Sequence characterization of the 5S ribosomal DNA and the internal transcribed spacer (ITS) region in four European Donax species (Bivalvia: Donacidae)

Abstract Background The whole repeat unit of 5S rDNA and the internal transcribed spacer (ITS) of four European Donax species were analysed. After amplifying, cloning and sequencing several 5S and ITS units, their basic features and their variation were described. The phylogenetic usefulness of 5S and ITS sequences in the inference of evolutionary relationships among these wedge clams was also investigated. Results The length of the 5S repeat presented little variation among species, except D. trunculus that differed from the rest of the Donax species in 170–210 bp. The deduced coding region covered 120 bp, and showed recognizable internal control regions (ICRs) involved in the transcription. The length of non-transcribed spacer region (NTS) ranged from 157 bp to 165 bp in Donax trunculus and from 335 bp to 367 bp in the other three species. The conservation degree of transcriptional regulatory regions was analysed revealing a conserved TATA-like box in the upstream region. Regarding ITS sequences, the four Donax species showed slight size differences among clones due to the variation occurring in the ITS1 and ITS2, except Donax variegatus did not display size differences in the ITS2. The total length of the ITS sequence ranged between 814 and 1014 bp. Resulting phylogenetic trees display that the two ribosomal DNA regions provide well-resolved phylogenies where the four European Donax species form a single clade receiving high support in nodes. The topology obtained with 5S sequences was in agreement with Donax evolutionary relationships inferred from several sequences of different nature in previous studies. Conclusions This is not only a basic research work, where new data and new knowledge is provided about Donax species, but also have allowed the authentication of these wedge clams and offers future applications to provide other genetic resources

Main structural features of the four sequenced mt genomes in this study.

<p>Main structural features of the four sequenced mt genomes in this study.</p

Presence of the <i>atp8</i> gene in the mitogenomes of the Veneroida order.

<p>Presence of the <i>atp8</i> gene in the mitogenomes of the Veneroida order.</p

Maps of the mitochondrial genomes of <i>Donax</i> species.

<p>Genome lengths are shown in the middle of each map, genes are all on “+” strand and NCR indicates the longest non-coding region.</p

Phylogenetic tree of the Veneroida order based on concatenated amino acids of 13 protein-coding genes.

<p>Numbers at the nodes correspond to Bayesian posterior probabilities (left), PhyloBayes posterior probabilities (middle) and ML bootstrap proportions (right). Dash indicates the difference in the position for <i>S</i>. <i>scabra</i> in the PhyloBayes phylogeny.</p

First complete female mitochondrial genome in four bivalve species genus <i>Donax</i> and their phylogenetic relationships within the Veneroida order

<div><p>Background</p><p>Four species of the genus <i>Donax</i> (<i>D</i>. <i>semistriatus</i>, <i>D</i>. <i>trunculus</i>, <i>D</i>. <i>variegatus</i> and <i>D</i>. <i>vittatus</i>) are common on Iberian Peninsula coasts. Nevertheless, despite their economic importance and overexploitation, scarce genetic resources are available. In this work, we newly determined the complete mitochondrial genomes of these four representatives of the family Donacidae, with the aim of contributing to unveil phylogenetic relationships within the Veneroida order, and of developing genetic markers being useful in wedge clam identification and authentication, and aquaculture stock management.</p><p>Principal findings</p><p>The complete female mitochondrial genomes of the four species vary in size from 17,044 to 17,365 bp, and encode 13 protein-coding genes (including the <i>atp8</i> gene), 2 rRNAs and 22 tRNAs, all located on the same strand. A long non-coding region was identified in each of the four <i>Donax</i> species between <i>cob</i> and <i>cox2</i> genes, presumably corresponding to the Control Region. The Bayesian and Maximum Likelihood phylogenetic analysis of the Veneroida order indicate that all four species of <i>Donax</i> form a single clade as a sister group of other bivalves within the Tellinoidea superfamily. However, although Tellinoidea is actually monophyletic, none of its families are monophyletic.</p><p>Conclusions</p><p>Sequencing of complete mitochondrial genomes provides highly valuable information to establish the phylogenetic relationships within the Veneroida order. Furthermore, we provide here significant genetic resources for further research and conservation of this commercially important fishing resource.</p></div

Comparison of non-coding regions (NCRs) within the four mt genomes.

<p>Comparison of non-coding regions (NCRs) within the four mt genomes.</p

Sampling details.

<p>Sampling details.</p